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12690
Effector Caspases and Substrates Antibody Sampler Kit
Primary Antibodies
Antibody Sampler Kit

Effector Caspases and Substrates Antibody Sampler Kit #12690

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Flow cytometric analysis of Daudi cells (blue) and MJ cells (green) using Lamin A/C (4C11) Mouse mAb (solid lines) or concentration-matched Mouse (G3A1) mAb IgG1 Isotype Control #5415 (dashed lines). Anti-mouse IgG (H+L), F(ab')2 Fragment (Alexa Fluor® 488 Conjugate) #4408 was used as a secondary antibody.
Simple Western™ analysis of lysates (1 mg/mL) from serum-starved HeLa cells treated with Staurosporine (1 uM, 3 hours) using PARP Antibody #9542. The virtual lane view (left) shows the target bands (as indicated) at 1:10 and 1:50 dilutions of primary antibody. The corresponding electropherogram view (right) plots chemiluminescence by molecular weight along the capillary at 1:10 (blue line) and 1:50 (green line) dilutions of primary antibody. This experiment was performed under reducing conditions on the Jess™ Simple Western instrument from ProteinSimple, a BioTechne brand, using the 12-230 kDa separation module.
Simple Western™ analysis of lysates (1 mg/mL) from Jurkat cells treated with Cytochrome C using Caspase-6 Antibody #9762. The virtual lane view (left) shows the target bands (as indicated) at 1:10 and 1:50 dilutions of primary antibody. The corresponding electropherogram view (right) plots chemiluminescence by molecular weight along the capillary at 1:10 (blue line) and 1:50 (green line) dilutions of primary antibody. This experiment was performed under reducing conditions on the Jess™ Simple Western instrument from ProteinSimple, a BioTechne brand, using the 12-230 kDa separation module.
Western blot analysis of extracts from HeLa cells (lane 1) or LMNB1 knock-out cells (lane 2) using Lamin B1 (D4Q4Z) Rabbit mAb #12586 (upper), and β-actin (D6A8) Rabbit mAb #8457 (lower). The absence of signal in the LMNB1 knock-out HeLa cells confirms specificity of the antibody for LMNB1.
Western blot analysis of extracts from Jurkat and A20 cells, untreated (-) or treated with Etoposide #2200 (25 μM, overnight; +), using Caspase-7 (D2Q3L) Rabbit mAb.
Western blot analysis of extracts from control HeLa cells (lane 1) or HeLa cells with an apparent in-frame truncation mutation in the gene encoding LMNA (lane 2) using Lamin A/C (4C11) Mouse mAb #4777 (upper) or α-actinin (D6F6) XP® Rabbit mAb #6487 (lower). The change in LMNA molecular weight in the mutated HeLa cells is consistent with an in-frame deletion.
After the primary antibody is bound to the target protein, a complex with HRP-linked secondary antibody is formed. The LumiGLO® is added and emits light during enzyme catalyzed decomposition.
After the primary antibody is bound to the target protein, a complex with HRP-linked secondary antibody is formed. The LumiGLO* is added and emits light during enzyme catalyzed decomposition.
Western blot analysis of extracts from NIH/3T3 cells, untreated or staurosporine-treated (1 µM), and Jurkat cells, untreated or etoposide-treated (25 µM), using PARP Antibody.
Western blot analysis of extracts from HeLa cells, transfected with 100 nM SignalSilence® Control siRNA (Fluorescein Conjugate) #6201 (-) or SignalSilence® Caspase-3 siRNA II (+), using Caspase-3 (8G10) Rabbit mAb and α-Tubulin (11H10) Rabbit mAb #2125. Caspase-3 (8G10) Rabbit mAb confirms silencing of caspase-3 expression, while the α-Tubulin (11H10) Rabbit mAb is used to control for loading and specificity of caspase-3 siRNA.
Western blot analysis of extracts from various cell lines, untreated or cytochrome c treated (1hr, 0.25 mg/ml in vitro) using Caspase-6 Antibody #9762 (upper) or β-Actin (D6A8) Rabbit mAb #8457 (lower).
Western blot analysis of extracts from various cell lines using Lamin B1 (D4Q4Z) Rabbit mAb.
Western blot analysis of extracts from various cell lines using Lamin A/C (4C11) Mouse mAb.
Western blot analysis of extracts from HeLa cells, transfected with 100 nM SignalSilence® Control siRNA (Fluorescein Conjugate) #6201 (-) or SignalSilence® Caspase-3 siRNA I (+), using Caspase-3 (8G10) Rabbit mAb and p42 MAPK Antibody #9108. Caspase-3 (8G10) Rabbit mAb confirms silencing of caspase-3 expression, while the p42 MAPK Antibody is used to control for loading and specificity of caspase-3 siRNA.
Western blot analysis of extracts from HeLa cells, untreated or treated with Staurosporine #9953 (1 μM, 3 hr), using Lamin B1 (D4Q4Z) Rabbit mAb.
Western blot analysis of extracts from THP-1 cells, untreated or treated with cycloheximide (CHX, 10 μg/ml, overnight) followed by TNF-α #8902 (20 ng/ml, 4 hr), using Lamin A/C (4C11) Mouse mAb.
Western blot analysis of HeLa (human) and NIH/3T3 (mouse) cell extracts, untreated and treated with 1 μM staurosporine (3 hr) in vivo, using Caspase-3 (8G10) Rabbit mAb.
Immunohistochemical analysis of paraffin-embedded human breast carcinoma using Lamin A/C (4C11) Mouse mAb.
Immunoprecipitation of cleaved caspase-3 from Jurkat cell extracts untreated (control) or treated with etoposide (25uM 5hrs) (apoptotic) using Caspase-3 (8G10) Rabbit mAb, and western probed with the same antibody.
Immunohistochemical analysis of paraffin-embedded human colon carcinoma using Lamin A/C (4C11) Mouse mAb.
Immunofluorescent analysis of normal rat brain using Lamin A/C (4C11) Mouse mAb (green) and MAP2 Antibody #4542 (red).
Confocal immunofluorescent analysis of HeLa cells using Lamin A/C (4C11) Mouse mAb (green). Actin filaments were labeled with DY-554 phalloidin (red).
Flow cytometric analysis of HeLa cells (green) using Lamin A/C (4C11) Mouse mAb (solid lines) or a concentration matched Mouse (G3A1) mAb IgG Isotype Control #5415 (dashed lines). Anti-mouse IgG (H+L), F(ab')2 Fragment (Alexa Fluor® 488 Conjugate) #4408 was used as a secondary antibody.
Inquiry Info.# 12690

Product Description

The Effector Caspases and Substrates Antibody Sampler Kit provides an economical means to evaluate the activation of effector (executioner) caspases. The kit contains enough primary antibody to perform at least four western blots per primary antibody.

Specificity / Sensitivity

Each antibody in the Effector Caspases and Substrates Antibody Sampler Kit recognizes endogenous levels of its respective target. Caspase-3 (8G10) Rabbit mAb recognizes full-length (35 kDa) and the large fragment (17/19 kDa) of caspase-3 resulting from cleavage at Asp175. Caspase-6 Antibody recognizes full length (35 kDa) and the small subunit (15 kDa) of caspase-6 resulting from cleavage at Asp193. Caspase-7 (D2Q3L) Rabbit mAb recognizes full-length (35 kDa) and the large subunit (20 kDa) of caspase-7 resulting from cleavage at Asp198. PARP Antibody recognizes full length (116 kDa) and the large fragment (89 kDa) of PARP1 resulting from caspase cleavage at Asp214. Lamin A/C (4C11) Mouse mAb recognizes full-length lamin A and lamin C proteins, and the larger fragments of lamin A (50 kDa) and lamin C (41 kDa) resulting from caspase cleavage. Lamin B1 (D4Q4Z) Rabbit mAb recognizes endogenous levels of total lamin B1 protein and a 25 kDa fragment resulting from caspase cleavage.

Source / Purification

Monoclonal antibodies are produced by immunizing animals with a synthetic peptide corresponding to amino-terminal residues adjacent to Asp175 in human caspase-3 protein, residues surrounding Pro158 of human caspase-7 protein, residues surrounding Leu118 of human lamin B1 protein, or with a recombinant fragment of human lamin A protein. Polyclonal antibodies are produced by immunizing animals with a synthetic peptide corresponding to residues surrounding the cleavage site of caspase-6 or the caspase cleavage site in PARP. Polyclonal antibodies are purified by protein A and peptide affinity chromatography.

Background

Apoptosis is a regulated physiological process leading to cell death. Caspases, a family of cysteine acid proteases, are central regulators of apoptosis. Caspase-3 (CPP-32, Apoptain, Yama, SCA-1), Caspase-6 (Mch2), and Caspase-7 (CMH-1, Mch3, ICE-LAP3) are effector caspases functioning in cellular apoptotic processes (1-6). Upon apoptotic stimulation, initiator caspases such as caspase-9 (ICE-LAP6, Mch6) are cleaved and activated (7). The activated upstream caspases further process downstream executioner caspases by cleaving them into activated large and small subunits, thereby initiating a caspase cascade leading to apoptosis (4,6,8-10).

PARP, a 116 kDa nuclear poly (ADP-ribose) polymerase, appears to be involved in DNA repair in response to environmental stress (11). This protein can be cleaved by many ICE-like caspases in vitro (1,12) and is one of the main cleavage targets of caspase-3 in vivo (10,13). In human PARP, cleavage occurs between Asp214 and Gly215, which separates the PARP amino-terminal DNA binding domain (24 kDa) from the carboxy-terminal catalytic domain (89 kDa) (10,12). PARP helps cells to maintain their viability; cleavage of PARP facilitates cellular disassembly and serves as a marker of cells undergoing apoptosis (14).

Lamins are nuclear membrane structural components that are important in maintaining normal cell functions, such as cell cycle control, DNA replication, and chromatin organization (15-17). Lamins have been subdivided into types A and B. Type-A lamins consist of lamin A and C, which arise from alternative splicing of the lamin A gene LMNA. Lamin A and C are cleaved by caspases into large (41-50 kDa) and small (28 kDa) fragments, which can be used as markers for apoptosis (18,19). Type-B lamins consist of lamin B1 and B2, encoded by separate genes (20-22). Lamin B1 is also cleaved by caspases during apoptosis (23).

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  4. Fernandes-Alnemri, T. et al. (1995) Cancer Res 55, 6045-52.
  5. Duan, H. et al. (1996) J Biol Chem 271, 1621-5.
  6. Lippke, J.A. et al. (1996) J Biol Chem 271, 1825-8.
  7. Li, P. et al. (1997) Cell 91, 479-89.
  8. Slee, E.A. et al. (1999) J Cell Biol 144, 281-92.
  9. MacFarlane, M. et al. (1997) J Cell Biol 137, 469-79.
  10. Nicholson, D.W. et al. (1995) Nature 376, 37-43.
  11. Satoh, M.S. and Lindahl, T. (1992) Nature 356, 356-8.
  12. Lazebnik, Y.A. et al. (1994) Nature 371, 346-7.
  13. Tewari, M. et al. (1995) Cell 81, 801-9.
  14. Oliver, F.J. et al. (1998) J Biol Chem 273, 33533-9.
  15. Dunbar, J.C. and Lu, H. (2000) Brain Res Bull 52, 123-6.
  16. Goldberg, M. et al. (1999) Crit Rev Eukaryot Gene Expr 9, 285-93.
  17. Yabuki, M. et al. (1999) Physiol Chem Phys Med NMR 31, 77-84.
  18. Rao, L. et al. (1996) J Cell Biol 135, 1441-55.
  19. Orth, K. et al. (1996) J Biol Chem 271, 16443-6.
  20. Biamonti, G. et al. (1992) Mol Cell Biol 12, 3499-506.
  21. Lin, F. and Worman, H.J. (1995) Genomics 27, 230-6.
  22. Pollard, K.M. et al. (1990) Mol Cell Biol 10, 2164-75.
  23. Chandler, J.M. et al. (1997) Biochem J 322 ( Pt 1), 19-23.

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